Project Summary/Abstract Approximately 30-50% of colorectal tumors carry activating missense mutations in KRAS. Late endosome- derived exosomes are often secreted from cancer cells and serve as mediators of cell-cell communication. Exosomes have been reported to be associated with tumor aggressiveness and metastasis. We have previously reported that the mutant KRAS status of colorectal cancer cells greatly affects the extracellular protein and RNA composition of exosomes. We also recently reported that sorting of Argonaute 2 (Ago2) and several Ago2-associated miRNAs to exosomes is controlled by KRAS-MEK-Erk signaling in colon cancer cells. Specifically, phosphorylation of Ago2 on serine 387 prevents Ago2 association with multivesicular endosomes (MVE) and sorting into exosomes. This finding identifies a molecular mechanism that has the potential to control the secretion of multiple miRNAs, miRNA-associated proteins, and potentially additional types of RNAs into exosomes. In this proposed project, I will identify the exact role of Argonaute 2 in sorting RNAs to exosomes, including determining what proportion of miRNAs are secreted in exosomes using this Ago2- controlled mechanism as well as whether there is selectivity in miRNA sorting. I will also determine whether other RNAs rely on Ago2 for their sorting into exosomes. Finally, I will investigate the functional consequence of Ago2-mediated RNA secretion from KRAS-dependent colon cancer cells on recipient cell gene expression and phenotypes both in vitro and in vivo. !